SYNTHESIS AND CHARACTERIZATION OF ...Co-supervisor: Assist. Prof. Dr. Serap Yalçın Azarkan August...

SYNTHESIS AND CHARACTERIZATION OF POLYETHYLENE GLYCOL

COATED MAGNETIC NANOPARTICLES AND THEIR USE FOR ANTI-CANCER

DRUG DELIVERY

A THESIS SUBMITTED TO

THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES

OF

MIDDLE EAST TECHNICAL UNIVERSITY

BY

MURAT ERDEM

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR

THE DEGREE OF MASTER OF SCIENCE

IN

BIOLOGY

AUGUST 2014

Approval of the thesis


COATED MAGNETIC NANOPARTICLES AND THEIR USE FOR ANTI-

CANCER DRUG DELIVERY

submitted by MURAT ERDEM in partial fulfillment of the requirements for the degree

of Master of Science in Biology Department, Middle East Technical University by,

Prof. Dr. Canan Özgen ____________________

Dean, Graduate School of Natural and Applied Sciences

Prof. Dr. Orhan Adalı _____________________

Head of Department, Biology

Prof. Dr. Ufuk Gündüz _____________________

Supervisor, Biology Dept., METU

Assist. Prof. Dr. Serap Yalçın Azarkan _____________________

Co-supervisor, Food Eng. Dept., AHİ EVRAN UNİ.

Examining Committee Members:

Assoc. Prof. Dr. Dilek (Şendil) Keskin _____________________

Engineering Sciences Dept., METU

Prof. Dr. Ufuk Gündüz _____________________

Biology Dept., METU

Assoc. Prof. Dr. Mayda Gürsel _____________________

Biology Dept., METU

Assoc. Prof. Dr.Çağdaş Devrim Son _____________________

Biology Dept., METU

Dr. Pelin Mutlu _____________________

METU Central Lab, Molecular Bio. and Biotech. R&D

Date: 28.08.2014

iv

I hereby declare that all information in this document has been obtained and

presented in accordance with academic rules and ethical conduct. I also declare

that, as required by these rules and conduct, I have fully cited and referenced all

material and results that are not original to this work.

Name, Last name: MURAT ERDEM

Signature:

V

ABSTRACT


COATED MAGNETIC NANOPARTICLES AND THEIR USE FOR ANTI-

CANCER DRUG DELIVERY

Erdem, Murat

M.S., Department of Biology

Supervisor: Prof. Dr. Ufuk Gündüz

Co-supervisor: Assist. Prof. Dr. Serap Yalçın Azarkan

August 2014, 73 pages

Although conventional chemotherapy is the most valid method to cope with cancer, it

has many drawbacks such as decrease in production of blood cells, inflammation of the

lining of the digestive tract, hair loss etc. Reasons for its side effects are that drugs used

in chemotherapy are distributed evenly within the body of a patient and cannot

distinguish the cancer cells from the healthy ones. To decrease the negative effects of

chemotherapeutic drugs and to increase their efficiency, many drug delivery systems

have been developed until now. Magnetic nanoparticles have an important potential for

cancer treatment. The most significant feature of magnetic nanoparticles is that they can

be manipulated for targeting to tumor side by application of external magnetic field.

Also, their small size and their capability to carry drug by surface modification are other

important characteristics for drug delivery.

Aim of this study is to synthesize folic acid conjugated, polyethylene coated magnetic

nanoparticles (FA-MNPs) and doxorubicin loaded formulation (Dox-FA-MNPs), and to

vi

investigate their cytotoxicity on HeLa and doxorubicin resistant HeLa cells. Magnetic

nanoparticles (MNPs), PEG coated MNPs and FA-MNPs were successfully synthesized

and characterized by one or more of TEM, FTIR, XRD, TGA and VSM analysis.

Doxorubicin (Dox) loading capacity of FA-MNPs and release profile of Dox from Dox-

FA-MNPs were investigated by spectrometer. Internalization of FA-MNPs and Dox-FA-

MNPs by HeLa cells were observed by purssian blue staining under light microscopy

and by using fluorescent property of Dox under fluorescent microscopy respectively.

Biocompatibility of FA-MNPs and antiproliferative effects of Dox-FA-MNPs on HeLa

and HeLa/Dox cells were analyzed by XTT cell proliferation assay.

The results show that synthesis of MNPs, PEG-MNPs and FA-MNPs were successfully

achieved. MNPs had a special shape and small size and also had supermagnetic behavior.

505 μM of 1724 μM Dox was loaded 500 μg/mL FA-MNPs and in 24 h, 68,35 μM Dox

was released from 500 μg/mL Dox-FA-MNPs. Also, drug release was increased in acidic

condition from Dox-FA-MNPs. Internalization experiments showed that FA-MNPs and

Dox-FA-MNPs were taken up by HeLa cells. FA-MNPs and Dox-FA-MNPs were given

to HeLa and HeLa/Dox cells for investigation of their cytotoxicities. Cell proliferation

assay results showed that Dox-FA-MNPs significantly decreased the proliferation of

HeLa cells when compared to FA-MNPs. However, Dox-FA-MNPs did not show same

effects on HeLa/Dox cells.

Despite that Dox-FA-MNPs could not overcome drug resistance of HeLa/Dox cells, they

effectively killed HeLa cells. As a result, FA-MNPs have found to be important for

carrying Dox.

Keywords: Cancer, HeLa, drug delivery system, magnetic nanoparticles

vii

ÖZ

POLİETİLEN GLİKOL KAPLI MANYETİK NANOPARÇACIKLARIN

SENTEZİ VE KARAKTERİZASYONU VE ANTİ-KANSER İLAÇ

TAŞINMASINDA KULLANIMI

Erdem, Murat

Yüksek Lisans, Biyoloji Bölümü

Tez Yöneticisi: Prof. Dr. Ufuk Gündüz

Ortak Tez Yöneticisi: Assist. Prof. Dr. Serap Yalçın Azarkan

Ağustos 2014, 73 sayfa

Geleneksel kemoterapi kanserle baş etmede en geçerli metot olmasına rağmen

kemoterapinin kan hücresi üretiminde azalma, sindirim sistemi zarının inflamasyonu,

saç dökülmesi gibi bir çok yan etkisi bulunmaktadır. Bu yan etkilerin nedeni

kemoterapide kullanılan ilaçların hastanın bedeninde eşit şeklide dağılması ve ilacın

kanser hücrelerini sağlıklı hücrelerden ayırt etmemesidir. Kemoterapötik ilaçların

olumsuz etkilerini azaltmak ve verimliliği artırmak için, şimdiye kadar birçok ilaç

taşıma sistemi geliştirilmiştir. Manyetik nanoparçacıklar kanser tedavisi için önemli bir

potansiyele sahiptir. Manyetik nanoparçacıkların en önemli özelliği, harici bir manyetik

alanın uygulanmasıyla tümör bölgesine hedeflenebilmesidir. Ayrıca, manyetik

nanoparçacıkların küçük boyutlu olmaları ve yüzey modifikasyonu ile ilaç taşıma

kapasitesi kazanabilmeleri, ilaç taşımacalığı için gerekli diğer önemli özellikleridir.

Bu çalışmanın amacı, folik asit bağlı, polietilen glikol kaplı manyetik nanoparçacıklar

(FA-MNP’ler) ve onların doksorubisin yüklü formülasyonunu (Dox-FA-MNP’ler)

viii

sentezlemek ve bu parçacıkların doksorubisine duyarlı HeLa ve doksorubisine dirençli

HeLa (HeLa/Dox) hücrelerindeki sitotoksisitelerini araştırmaktır. Manyetik

nanoparçacıklar (MNP’ler), PEG kaplanmış MNP’ler (PEG-MNP’ler) ve FA-MNP’ler

başarılı bir şekilde sentezlenmiş ve TEM, FTIR, XRD, TGA ve VSM analizlerinden biri

ya da daha fazlası ile karakterize edilmiştir. FA-MNP’lerin doksorubisin (Dox) yükleme

kapasitesi ve Dox-FA-MNP’lerin Dox salma profili spektrometre ile incelenmiştir. FA-

MNP ve Dox-FA-MNP’lerin HeLa hücreleri tarafından hücre içine alımı sırasıyla ışık

mikroskobu altında purssian mavi boyama yöntemiyle ve Dox’un floresan özelliği

kullanılarak floresan mikroskobu altında gözlenmiştir. HeLa ve HeLa/Dox hücrelerine

FA-MNP’lerin biyouyumluluğu ve Dox-FA-MNP’lerin bu hücrelerin çoğalmasına karşı

etkileri, XTT-hücre proliferasyon deneyi ile analiz edilmiştir.

Sonuçlar MNP’lerin, PEG-MNP’lerin ve FA-MNP’lerin sentezinin başarılı bir şekilde

gerçekleştirldiğini göstermektedir. İncelemeler, MNP’lerin özel bir şekle sahip

olduğunu, küçük boyutlu olduğunu ve süperparamanyetik davranış sergilediği

göstermiştir. 500 μg/mL FA-MNP’ye 505 μM Dox’un yüklendiği ve 24 saat içinde,

68,35 μM Dox’un Dox-FA-MNP’lerden salımdığı görülmüştür. Aynı zamanda, ortamın

asitliliği 500μg/mL Dox-FA-MNP’lerin ilaç salımını arttırmıştır. Mikrosopi ile yapılan

gözlemler, FA-MNP’lerin ve Dox-FA-MNP’lerin HeLa hücreleri tarafından alındığını

göstermiştir. FA-MNP’lerin ve Dox-FA-MNP’lerin sitotoksisite analizleri HeLa ve

HeLa/Dox hücreleri ile yapılmıştır. Hücre çoğalma analizi sonuçları FA-MNPS ile

karşılaştırıldığında–Dox-FA-MNP’lerin HeLa hücrelerinin çoğalmasını önemli ölçüde

azalttığını göstermiştir. Ancak, Dox-FA-MNP’ler HeLa/Dox hücreleri üzerinde aynı

etkiyi gösterememiştir.

Dox-FA-MNP’ler HeLa/Dox hücrelerinin ilaç dirençliliğinin geri çevrilmesinde etkili

olmamasına rağmen, HeLa hücrelerini etkili bir şekilde öldürmüşlerdir. Sonuç olarak,

FA-MNP’lerin Dox taşıması için önemli olduğu bulunmuştur.

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Anahtar Sözcükler: Kanser, HeLa, ilaç taşıma sistemi, manyetik nanoparçacık

x

To My Sisters: Fatma and Safiye,

xi

ACKNOWLEDGEMENTS

I would like to express my special appreciation and thanks to my supervisor Prof. Dr.

Ufuk Gündüz for her endless support, priceless advice, and immense knowledge

throughout the thesis.

I would like to thank to my co-supervisor Assist. Prof. Dr. Serap Yalçın Azarkan for her

support and suggestions.

I would like to express the deepest appreciation to the members of examining

committee, Assoc. Prof. Dr. Dilek Şendil Keskin, Assoc. Prof Dr. Mayda Gürsel, Assoc.

Prof Dr. Çağdaş Devrim Son and Dr. Pelin Mutlu.

I am deeply thankful to Çağrı Urfalı, Ayça Nabioğlu, Gözde Ünsoy, Maryam Persian

and Negar Taghavi for their experience and advices. Special thanks to my everlasting

lab-mate Aktan Alpsoy for his valuable friendship, encouragement and support.

I am thankful to previous members of Lab206 Neşe Çakmak, Esra Kaplan, Ahu İzgi,

Tuğba Keskin, Gülistan Tansık, Zelha Nil, Okan Tezcan, Burcu Özsoy, Gülşah Pekgöz

and Çiğdem Şener.

This study is supported by METU Research Fund (Grant ID: BAP-07-02-2012-101).

xii

TABLE OF CONTENTS

ABSTRACT ...................................................................................................................... v

ÖZ ................................................................................................................................... vii

ACKNOWLEDGEMENTS ............................................................................................ xi

TABLE OF CONTENTS ............................................................................................... xii

LIST OF TABLES ........................................................................................................ xvi

LIST OF FIGURES ...................................................................................................... xvii

LIST OF ABBREVIATIONS ......................................................................................... xx

CHAPTERS

1 INTRODUCTION ..................................................................................................... 1

1.1 Cancer ................................................................................................................. 1

1.2 Cervical Cancer .................................................................................................. 2

1.3 Treatment Options for Cervical Cancer .............................................................. 2

1.3.1 Surgery ........................................................................................................ 2

1.3.2 Radiation Therapy ....................................................................................... 3

1.3.3 Chemotherapy ............................................................................................. 4

1.3.4 Side effects of Chemotherapy ..................................................................... 5

1.4 Targeted Therapy ................................................................................................ 7

1.5 Drug Delivery Systems ....................................................................................... 7

1.6 Magnetic Nanoparticles ...................................................................................... 9

1.6.1 Biomedical Applications of Magnetic Nanoparticles ............................... 10

xiii

1.6.2 Magnetic Nanoparticles for Drug Delivery ............................................... 11

1.6.3 Structure of Magnetic Nanoparticles ........................................................ 12

1.6.4 Polyethylene Glycol (PEG) in Drug Delivery .......................................... 13

1.6.5 Folic Acid Directed Drug Delivery ........................................................... 15

1.7 Synthesis Methods of Magnetic Nanoparticles ................................................ 16

1.8 Objective of the Study ...................................................................................... 18

2 MATERIALS AND METHODS ............................................................................. 19

2.1 Materials for Magnetic Nanoparticle Synthesis ............................................... 19

2.2 Synthesis of Magnetic Nanoparticles ............................................................... 19

2.3 Synthesis of PEG Coated Magnetic Nanoparticles .......................................... 20

2.4 Folic Acid Modification of PEG-MNP ............................................................ 20

2.5 Drug Loading.................................................................................................... 21

2.6 Drug Release .................................................................................................... 21

2.7 Cell Culture ...................................................................................................... 22

2.7.1 Cell Line and Culture Condition ............................................................... 22

2.7.2 Subculturing .............................................................................................. 22

2.7.3 Cell Freezing ............................................................................................. 23

2.7.4 Cell Thawing ............................................................................................. 23

2.7.5 Cell Counting ............................................................................................ 24

2.8 Internalization of Nanoparticles ....................................................................... 24

2.8.1 Detection of Internalized Nanoparticles by Purssian Blue Staining ......... 25

2.8.2 Detection of Internalized Nanoparticles by Fluorescent Microscopy ....... 26

xiv

2.8.3 Cell Proliferation Assay ............................................................................ 26

2.9 Statistical analysis............................................................................................. 28

3 RESULTS AND DISCUSSION .............................................................................. 31

3.1 Chemical Characterization ............................................................................... 31

3.1.1 Chemical Characterization of MNPs ......................................................... 31

3.1.1.1 Transmission Electron Microscopy of MNPs ....................................... 32

3.1.1.2 X-Ray Diffraction .................................................................................. 34

3.1.1.3 Fourier Transform Infrared Spectroscopy of MNPs .............................. 35

3.1.1.4 TGA (Thermal Gravimetric Analysis) of MNPs ................................... 36

3.1.1.5 VSM (Vibrating Sample Magnetometer) .............................................. 37

3.1.2 Chemical Characterization of PEG-MNP ................................................. 38

3.1.2.1 TEM Characterization of PEG-MNP .................................................... 38

3.1.2.2 Fourier Transform Infrared Spectroscopy (FTIR) of PEG-MNP .......... 40

3.1.2.3 Thermal Gravimetric Analysis (TGA) .................................................. 41

3.1.3 Chemical Characterization of FA-MNPs .................................................. 43

3.1.3.1 Fourier Transform Infrared Spectroscopy of FA-MNPs ....................... 43

3.2 Drug Loading .................................................................................................... 44

3.3 Drug Release ..................................................................................................... 46

3.4 Cell Culture Studies .......................................................................................... 47

3.4.1 Development of Drug Resistant Cell Line ................................................ 47

3.4.1.1 Internalization of Nanoparticles ............................................................ 49

3.4.1.2 Detection of FA-MNPs in HeLa Cells by Purssian Blue Staining ........ 49

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3.4.1.3 Detection of Dox-FA-MNPs in HeLa Cells by Fluorescent Microscopy

………………………………………………………………………...52

3.4.2 Antiproliferative Activity of Nanoparticles .............................................. 54

4 CONCLUSION ........................................................................................................ 57

REFERENCES ................................................................................................................ 59

APPENDICES

A BUFFERS AND SOLUTIONS ................................................................................... 65

B ATR AND FTIR SPECTRA ....................................................................................... 67

C CYTOTOXITY OF FA-MNPs ON HeLa AND HeLa/Dox CELL LINES ................ 69

D STANDARD CURVE OF DOXORUBICIN ............................................................. 71

E CONCENTRATION OF DOXORUBICIN LOADED TO FA-MNPs ....................... 73

xvi

LIST OF TABLES

TABLES

Table 1.1: Comperassion of MNP Synthesis Methods (Lu et al., 2007)......................... 17

Table 3.1: Concentration of doxorubicin loaded to FA-MNPs. ...................................... 45

Table 3.2: Concentration of released doxorubicin from Dox-FA-MNPs in different

solution and different pH Values. ................................................................................... 47

xvii

LIST OF FIGURES

FIGURES

Figure 1.1: Drug delivery systems for cancer treatment (Lembo & Cavalli, 2010). ........ 9

Figure 1.2: Biomedical applications of magnetic nanoparticles (Umut, 2013). ............. 10

Figure 1.3: Schematic representation of Magnetic drug delivery system under the

influence of external magnetic field (Mody et. al, 2013). .............................................. 12

Figure 1.4: General structure of a magnetic nanoparticle (Pereyra & Claudia, 2013). ... 13

Figure 1.5: Structure of polyethylene glycol (Sigma-Aldrich, n.d.) ............................... 14

Figure 1.6: Structure of folic acid (Sigma-Aldrich, n.d.-a). ............................................ 15

Figure 1.7: Representative picture of the folate receptor-mediated endocytosis pathway

(Ashley et. al, 2011). ....................................................................................................... 16

Figure 2.1: Representative picture of cell seeded 98 well plate. Gray wells represent cell

seeded ones. ..................................................................................................................... 27

Figure 2.2: Representative picture of drug dilution in 96 well plate. ............................. 28

Figure 3.1: TEM images of MNPs (a): in Ethanol, b): in Hexane) ................................. 33

Figure 3.2: X-Ray powder diffraction (XRD) patterns of synthesized iron oxide (OL-

Fe3O4) nanoparticles. ....................................................................................................... 34

Figure 3.3: FTIR spectrum of MNPs. ............................................................................. 35

Figure 3.4: Thermal gravimetric analyses of MNPs. ...................................................... 36

Figure 3.5: Vibrating sample magnetometer (VSM) results show magnetization and

demagnetization curves of MNPs (55 emu/g) at 37˚C. ................................................... 37

Figure 3.6: TEM images of PEG- MNPs. ....................................................................... 39

Figure 3.7: FTIR spectrum of PEG-MNPs. .................................................................... 40

Figure 3.8: Schematic representation of PEG coated MNPs (Yang et. al, 2010). .......... 41

Figure 3.9: Thermal gravimetric analyses of PEG- MNPs. ............................................ 42

xviii

Figure 3.10: FTIR spectrum of FA-MNPs. ..................................................................... 43

Figure 3.11: Percentages of doxorubicin loaded to FA-MNPs in different concentrations

of doxorubicin. ................................................................................................................ 44

Figure 3.12: Released doxorubicin concentration from Dox-FA-MNPs in acetate buffers

with different pH values. ................................................................................................. 46

Figure 3.13: Cell proliferation profile of HeLa cells treated with increase in

Doxorubicin concentration .............................................................................................. 48

Figure 3.14: Cell proliferation profile of HeLa/Dox cells treated with increase in

Doxorubicin concentration .............................................................................................. 49

Figure 3.15: Images of HeLa cells treated with 0 μM (a), 25 μg (b), 50 μg (c), 100 μg

(d), 150 μg (e) and 200 μg FA-MNPs (f). ....................................................................... 50

Figure 3.15: Images of HeLa cells treated with 0 μM (a), 25 μg (b), 50 μg (c), 100 μg

(d), 150 μg (e) and 200 μg FA-MNPs (f) (Continued). ................................................... 51

Figure 3.16: Images of HeLa cells treated with doxorubicin (a), 50 μg/mL (b), 150

μg/mL (c) and 200 μg/mL (d) Dox-FA-MNPs. .............................................................. 53

Figure 3.17: Antiproliferative effects of FA-MNPs and Dox-FA-MNPs on HeLa cell

line. Results were obtained from three independent experiments, represented as mean

SEM (*** Results were significant with a p<0.05). ....................................................... 54

Figure 3.18: Antiproliferative effects of FA-MNPs and Dox-FA-MNPs on HeLa/Dox

cell line. Results were obtained from three independent experiments, represented as

mean SEM (*** Results were significant with a p<0.05)............................................ 55

Figure B. 1: ATR Spectrum of PEGmonooleate ............................................................. 67

Figure B. 2: FTIR Spectrum of Folic Acid. .................................................................... 68

Figure C. 1: Antiproliferative effects of FA-MNPs on HeLa cell line. Results were

obtained from three independent experiments. represented as mean SEM. ................ 69

Figure C. 2: Antiproliferative effects of FA-MNPs on HeLa/Dox cell line. Results were

obtained from three independent experiments. represented as mean SEM. ................ 70

xix

Figure D. 1: Representative standard curve of Doxorubicin. .......................................... 71

Figure E. 1: Concentrations of Doxorubicin that was loaded to FA-MNPs in different

concentrations of Doxorubicin. ....................................................................................... 73

xx

LIST OF ABBREVIATIONS

ATR Attenuated Total Reflection

DCC Drug delivery system

DMSO Dimethyl sufoxide

Dox Doxorubicin

Dox-FA-

MNP

Dox loaded magnetic nanoparticle

FA-MNP Folic acid conjugated magnetic nanoparticle

Fe(acac)3 Iron(III) acetylacetonate

FTIR Fourier Transform Infrared Spectroscopy

HeLa/Dox 500 nM Doxorubicin-resistant HeLa cell line

IC50 Inhibitory concentration-50

MNP Magnetic nanoparticle

MRI Magnetic resonance imaging

MDR Multidrug resistance

MDR1 Multidrug resistance protein 1

OL-Fe3O4 Oleic acid and oleylamine coated Fe3O4

PBS Phosphate-buffered saline

PEG Poly ethylene glycol

PEG-MNP PEG coated magnetic nanoparticle

RES Reticuloendotelial system

TEM Transmission electron microscopy

TGA Thermal gravimetric analysis

Tris Tris(hydroxymethyl)aminomethane

xxi

VSM Vibrating sample magnetometer

XTT 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-

Carboxanilide

1

CHAPTER 1

1 INTRODUCTION

1.1 Cancer

Cancer is defined as a group of more than 100 diseases which has two main

characteristics, uncontrolled growth of the cells and the ability of these cells to migrate

from the original site and to spread to distant sites (American Cancer Society, 2013b).

There are two main groups of genes that trigger the development of cancer. Normally,

these genes have roles in the regulation of cell cycle, cell growth and cell division. First

group of genes is proto-oncogenes that promote the cell to divide while the other gene

group is called tumor-suppressor genes which have a role in inhibition of cell

proliferation. By working together both gene groups provide cells to grow and to divide

in regulated manner, so the cells function normally and meet their responsibilities for

the healthy body. If mutations occur in proto-oncogenes and tumor-suppressor genes,

proto-oncogenes turn into oncogenes and tumor-suppressor genes lose their function.

Oncogenes cause cells to proliferate irregularly while inactivated tumor-suppressor

genes cannot control the cell division. Thus, mutations occurred in these gene groups

are the main causes for the abnormal cell proliferation observed in cancers. Unlike

normal cell acts, cancer cells grow and proliferate irregularly. Then, they colonize in

healthy tissues and organs. Finally they metastasize the body (National Institutes of

Health, 2007).

2

1.2 Cervical Cancer

According to GLOBOCAN 2013, cancer incidence among women was recorded as

6.0447 million in worldwide in 2008. Cervical cancer is accounted for 275 000 deaths

worldwide in 2008. Cervical cancer is the third common cancer types globally

occurring among women.

Cervical cancer occurs in the cervix which is the organ located between the uterus and

vagina. It is usually a slow-growing cancer that may not have symptoms but can be

found with regular Pap tests (a procedure in which cells are scraped from the cervix and

looked at under a microscope). Cervical cancer is almost always caused by human

papillomavirus infection (National Cancer Institute, 2013a).

1.3 Treatment Options for Cervical Cancer

Cervical cancer has some different treatment options which are the currently used

treatments and tested treatments in laboratories and clinical trials. Standard treatments

are surgery, radiotherapy and chemotherapy. Developing methods are research studies

to improve current treatments or obtain information on new treatments for cervical

cancer. If the research studies on treatment of cervical cancer show better results than

the current treatments, the new treatment could be the standard treatment (National

Cancer Institute, 2013b).

1.3.1 Surgery

The most common method for the treatment of many different cancer types is surgery

which is the removal of affected area and surrounding tissue. Three main types of

http://www.cancer.gov/Common/PopUps/popDefinition.aspx?id=45961&version=Patient&language=English

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surgery which are applied to cervical cancer are radical trachelectomy, hysterectomy

and pelvic exenteration. Firstly, radical trachelectomy is the operation which includes

the removal of the cervix, surrounding tissue and the upper part of the vagina but not

the womb. Secondly, in hysterectomy, the cervix and womb are removed while

depending on the stage of the cancer, the ovaries and fallopian tubes may also be

necessary to be removed. Finally, pelvic exenteration is a major operation which

removes the cervix, vagina, womb, bladder, ovaries, fallopian tubes and rectum (Health

News Stories, 2013).

After surgery, patients could experience some side effects. First one is urination

problem. Women may have feeling that they could not urinate. Secondly, the removal

of ovary might cause women to enter menopause. Thirdly, if during surgery, lymph

nodes are removed, swelling occurs in legs due to the malfunction in lymphatic

systems. Also, women may experience physical and emotional changes, so their feeling

about sex may be affected (Cancer Council Victoria, 2013).

1.3.2 Radiation Therapy

Radiation therapy provides cervical cancer patients high-energy rays to destroy cancer

cells. Radiation therapy can be applied cervical cancer patients in any stages. Instead of

surgery, radio therapy may be used on patients in early stage of cervical cancer. Also, it

could be applied to kill any cancer cells left in the area after surgery. Radiation therapy

with chemotherapy may be used to kill cancer that spread out side of the cervix.

Radiation therapy could be applied to patients internally, externally or together. In

external radiation therapy radiation is focused on patients’ pelvis or other cancerous

areas while in internal radiation therapy called brachy therapy, a radioactive substance

http://www.cancer.gov/Common/PopUps/popDefinition.aspx?id=CDR0000044971&version=Patient&language=English

http://www.cancer.gov/Common/PopUps/popDefinition.aspx?id=CDR0000045151&version=Patient&language=English

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is given to patients via a narrow cylinder located inside their vagina (National Cancer

Institute, 2012).

1.3.3 Chemotherapy

Chemotherapy is the treatment of cancer by anti-neoplastic drugs. These drugs also

called chemotherapeutic drugs are used to destroy rapidly dividing cancer cells

(Mandal, 2014). Chemotherapy could be used alone or it can be applied to patients with

other cancer treatments, such as radiation therapy or surgery. Chemotherapy could

reduce the size of a tumor before the application of other therapies. Also, it can kill

cancer cells that are left after surgery or that escape from radiation therapy.

Chemotherapy increases the influence of radiation therapy on cancer. Moreover,

chemotherapy aims to prevent recurrence of cancer and metastasis of cancer (Wedro,

2014).

Chemotherapeutic drugs can be applied in many ways. They can be orally administered

as a pill. They can be injected into the muscle or fat tissue or they can be intravenously

given to cancer patients. Moreover, these drugs can be applied to the skin. Also, they

can be injected directly into a body cavity. Chemotherapeutic drugs can be divided into

several groups depending on mechanisms of their action, their chemical structure, and

their relationship with another drug. These groups are alkylating agents,

antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors and

corticosteroids. Alkylating agents prevent the proliferation of cancer cells by binding

covalently their alkyl groups to DNA molecules. These agents work effectively in all

phases of the cell cycle by damaging DNA. Sometimes they can cause acute leukemia

because their long-term usage could be harmful to the bone marrow. Antimetabolites

inhibit DNA and RNA synthesis by substituting nucleic acids. These agents are toxic to

http://en.wikipedia.org/wiki/Radiation_therapy

http://en.wikipedia.org/wiki/Surgery

5

cells and prevent the cell division at the S phase. Anti-tumor antibiotics also known as

anthracyclines interfere with enzymes that have roles in DNA replication. They are

effective in all phases of the cell cycle. Use of these agents in high doses can cause

permanent damage to the heart. Topoisomerase inhibitors prevent topoisomerases from

helping separation of DNA strands during replication. Treatment with these inhibitors

could cause a second cancer such as acute myelogenous leukemia. Mitotic inhibitors

are generally plant alkaloids acquired from natural products. These drugs can inhibit

mitosis by preventing enzymes that have roles in production of proteins required for cell

division. Although they are mainly effective at M phase of the cell cycle, these agents

could kill cells in any phases. These drugs can damage peripheral nerve. Corticosteroids

are hormones naturally produced in body and hormone-like drugs which can be helpful

in killing cancer cells. They are used to treat lymphoma, leukemia, and multiple

myeloma.

Chemotherapy uses more than one drug together to treat cervical cancer because

combination of drugs has more effective in killing cervical cancer cells. Drugs mostly

used in cervical cancer are cisplatin, carboplatin, paclitaxel, topotecan and gemcitabine.

Cisplatin and carboplatin are platinum drugs but sometimes they are classified as

alkylating agents because even they do not have alkyl group, they affect cancer cells as

alkylating agents do. Paclitaxel belongs to the family of mitotic inhibitors. Topotecan is

a topoisomerase inhibitor while gemcitabine is in the group of antimetabolites

(American Cancer Society, 2013a).

1.3.4 Side effects of Chemotherapy

Chemotherapy mainly affects the rapidly growing and dividing cells. This feature

defines cancerous cells, but it can be seen in healthy cells that also actively grow and

http://www.cancer.org/ssLINK/cisplatin

http://www.cancer.org/ssLINK/carboplatin

http://www.cancer.org/ssLINK/paclitaxel

http://www.cancer.org/ssLINK/topotecan

http://www.cancer.org/ssLINK/gemcitabine

6

proliferate. Such cells are found in the blood, mouth, intestines, and hair. Chemotherapy

causes the side effects when it causes damages to these normal cells because

chemotherapy cannot distinguish the cancer cells from the healthy ones. These side

effects cause patients to suffer fatigue (tiredness), pain in different parts of body, sores

in the mouth and throat, diarrhea, nausea and vomiting, blood disorders due to decrease

in blood cell number, nerve damage, changes in thinking and memory, sexual and

reproductive issues, appetite loss and hair loss. Also, chemotherapy causes long-term

side effects. After chemotherapy treatment ends, most of draw backs do not sustain

while, some effects may continue, or appear back, or later. For example, particular

chemotherapeutics are responsible for persistent damage to some organs such as the

heart, lung, liver, kidneys, or reproductive system. Moreover, it takes months and years

for cognitive functions work properly after treatment. Changes in nervous system also

can occur after treatment, and patients who were treated with chemotherapy in

childhood could be exposed to late effects. The possibility of occurrence of second

cancer is high for patients that beat cancer before (Schuchter, 2014).

The main problem that makes chemotherapy less effective in cancer treatment is the

drug resistance. Cancer cells could be intrinsically resistant to chemotherapy drugs or

initially sensitive cancer cells can become drug resistant during application of

chemotherapy. Acquired resistant cancer cells become not only resistant to a certain

chemotherapeutic drug but also resistant to chemically different kinds of drugs. Cancer

cell can become resistant to chemotherapy drug in many ways, by elevating drug efflux

and reducing drug influx; drug inactivation; alterations in drug target; processing of

drug-induced damage; and evasion of apoptosis (Longley & Johnston, 2005). From

them, the most important reason for multidrug resistance is that P-glycoprotein as

product of MDR1 gene is expressed in a high level. This transporter protein reduces

intracellular drug concentration by pumping out of the drug, so it causes a reduce in the

cytotoxic effect of drug (Di Pietro et al., 1999).

http://www.cancer.net/node/24571

7

1.4 Targeted Therapy

Targeted therapy means that a drug is designed to kill only cancer cells; but not healthy

cells, by targeting a specific protein only found in cancer cells, not in normal cells. This

therapy differentiates conventional chemotherapy by their selective cytotoxicity to

cancer cells (Wu, Chang, & Huang, 2006). Determination of antibody that selectively

binds cancer specific antigen gains importance in targeting a chemotherapy drug to

cancer cells. For instance, anti-CD33–calicheamicin is one of the antibody-drug

conjugates. It is composed of anti-CD33 antibody that specifically binds CD33 a

trasmembrane protein overexpressed in acute myeloid leukemia and calicheamicin

which attaches DNA and causes strand scission. Anti-CD33–calicheamicin is accepted

to be used to treat acute myeloid leukemia (Chari, 2008).

1.5 Drug Delivery Systems

Drug delivery system (DDS) is a platform that introduces a chemotherapy agent to body

and enhance the influence and safety of the drug in a way in which it can determine how

long, how fast and where drug release occurs in the body (Attama, Momoh, & Builders,

2012). Conventional chemotherapeutic drugs are spread out evenly within the body; as

a result, they have effect not only on cancer cells but also on healthy cells. Due to this

even distribution of these drugs, cancerous tissue gets limiting dose of the drugs.

Increase in the dose of the drug to kill more cancer cells causes the excessive toxicity to

body (Cho, Wang, Nie, Chen, & Shin, 2008). To solve these problems, DDSs have been

designed to increase influence of chemotherapy drug and decrease their toxicity by not

only providing increment of drug accumulation in tumor site but also lowering quantity

of drug distributed to healthy parts of body (Needham & Dewhirst, 2001).

8

The most important feature of DDSs is that drugs in the systems are released in a

controlled manner. Until now, many new DDSs have been developed such as capsules,

polymers, liposomes, microparticles and nanoparticles. These systems must carry some

necessary properties which include biocompatibility, biodegradability and a targeted

biodistribution at desired region supplying the therapeutic agents for a long time

(Mainardes & Silva, 2004).

A drug can be targeted to a specific tissue in different manners. Passive targeting is that

carrier directs the drug to a specific tissue via its main properties, such as size or

lipophilicity. When passive targeting is combined with the property of nanocarriers

which is being in small size, it provides drugs or nanocarriers to penetrate into tumor

region and accumulate in this region in which vasculature is disrupted and is leaky

(Figure 1.1) (Lembo & Cavalli, 2010). Active targeting is a targeting mechanism in

which drugs or carriers are targeted to particular cell types via recognitions of cell

specific proteins by ligands or molecules bound to these drugs or

carriers. Physical targeting means that drugs and carrier systems are distributed by

external influences, such as magnetic field or heat (Neuberger, Schöpf, Hofmann,

Hofmann, & von Rechenberg, 2005)

9

Figure 1.1: Drug delivery systems for cancer treatment (Lembo & Cavalli, 2010).

In many different size and structures, nano drug delivery systems offer techniques to

direct and release large amount therapeutic compounds in specific regions. New

developments in surface modification and production methods make nanoparticles to

get attentions in drug delivery technology. Many different types of these nanostructures

such as simple metal core or complex lipid-polymer constructions become functional in

many ways to work as drug carriers for diversity of situations (Willis, 2004).

1.6 Magnetic Nanoparticles

Magnetic nanoparticles (MNPs) as their name implies are in nanoscale size and can be

manipulated by an external magnetic field. Iron oxide (Fe3O4) MNPs around 20 nm in

10

size are defined as superparamagnetic. Because superparamagnetic particles such as

Fe3O4 are magnetized under magnetic field and they lose any magnetism in the absence

of magnetic field, they get many attentions on their applications for biomedicine (Berry

& Curtis, 2003).

1.6.1 Biomedical Applications of Magnetic Nanoparticles

For diagnostic purposes, MNPs have been used in in vivo for decades. MNPs are

involved in many different biomedical applications due to their distinctive properties

that include low toxicity, magnetic behavior and small size (Berry & Curtis, 2003).

MNPs have been applied in magnetic resonance imaging (MRI) for diagnosis of

diseases, in targeted therapy (hyperthermia) and in drug delivery (Figure 1.2) (Umut,

2013).

Figure 1.2: Biomedical applications of magnetic nanoparticles (Umut, 2013).

11

MRI has been used to differentiate one tissue from another tissue through high

resolution contrast. MRI systems use contrast agents to increase their diagnostic

purposes. Due to their 3–10 nm sizes, paramagnetic ion chelates and ferromagnetic or

superparamagnetic nanoparticles have been used as MR contrast agents in clinical

diagnosis (Ito, Shinkai, Honda, & Kobayashi, 2005).

In magnetic hyperthermia, MNPs are given to cancer patient and are directed and easily

delivered to tumor tissue by application of external magnetic field. Magnetized MNPs

in tumor region lose their magnetic energy as heat by changing external magnetic field.

This magnetic energy loss in form of heat increases the temperature in tumor tissue and

as a result cause cancer cells to die (Gkanas, 2013).

In magnetic targeting, a chemotherapy agent is loaded to a magnetic MNP. Then, drug-

bound MNPs are given to the body, and accumulated in the desired region by

application of an external magnetic field. In the targeted region, drugs separate from

MNPs or cause a local effect (Arruebo, Fernández-pacheco, Ibarra, & Santamaría,

2007).

1.6.2 Magnetic Nanoparticles for Drug Delivery

MNPs have become popular in drug delivery systems because these particles are

targeted by magnetic field, which is physical targeting. Therapeutic agents that are

delivered to the specific area are either bound to the surface of MNPs or encapsulated

by the magnetic nanoparticulate carriers. These agent-loaded MNPs aggregate at the

particular desired site by application of an external magnetic field (Figure 1.3) (Mody

et. al, 2013). Drug can be released from MNPs by simple diffusion or by mechanisms

that involve enzymatic activity or by changes of physiological conditions such as pH,

12

osmolarity, or temperature. Also, release of drug from the drug-linked MNPs can occur

magnetically (Arruebo et. al, 2007).

Figure 1.3: Schematic representation of Magnetic drug delivery system under the

influence of external magnetic field (Mody et. al, 2013).

1.6.3 Structure of Magnetic Nanoparticles

MNPs possess many important structures for different applications of biomedicine.

Although they have different structures, all MNPs have to own some fundamental

properties to function as DDS. Moreover, these particles need to have enough magnetic

properties to accumulate at the desired cells in the presence of a magnetic force. Also,

the architecture of MNPs needs to be compatible enough with biological entity to apply

to living cells or organisms. MNPs have structure in which a magnetic core is

composed of magnetite (Fe3O4) or maghemite (Fe2O3) and synthetic polymers protect

MNPs and make them biologically functional by coating the magnetic core. Sometimes,

13

for drugs or gene vectors to attach the MNPs, they are bound with specific organic

linkers (Figure 1.4) (Pereyra & Claudia, 2013).

Figure 1.4: General structure of a magnetic nanoparticle (Pereyra & Claudia,

2013).

1.6.4 Polyethylene Glycol (PEG) in Drug Delivery

Polyethylene glycol (PEG) has been a good choice for many years as a drug carrier

because it is biocompatible, least toxic and antigenic and high soluble in common

solvents or in water. Numerous agents and biological materials like proteins and

enzymes have been link to the ends of PEG chains and they can stay biological and

enzymatic active (Won, Chu, & Lee, 1998). The structure of PEG is given in Figure 1.5.

14

Figure 1.5: Structure of polyethylene glycol (Sigma-Aldrich, n.d.)

Phagocytes eliminate microparticulate carriers from the circulatory system by

opsonization, and surface modification of these carriers with PEG delays opsonization.

Also, PEG decreases the accumulation of microparticulate carriers by reticuloendotelial

system (RES). These 100-200 nm drug carriers with long circulation time efficiently

accumulate at tumor sites due to the enhanced permeability and retention effect

(Torchilin, 2001).

PEG also has an important role to functionalize the surface groups of dendrimers. As a

result, addition of PEG to dendrimers not only increases the water solubility of them but

also limits their immunogenicity. Moreover, PEG prevents derivatives of PEGylated

dendrimer in the biological condition by increasing their time spent in circulation,

which is vital feature for drug delivery systems. Also, PEG coating improves solubility

of hydrophobic compounds (Sideratou, Kontoyianni, Drossopoulou, & Paleos, 2010).

To function as delivery systems, multifunctional MNPs need to be designed carefully

with important properties. Their surface would protect them from the rapid elimination

by the RES after intravenous administration. They need to have drug-loading capacity

without changing the physical features of the MNP architecture or magnetization

property of the core material, and ability to conjugate MNPs to a targeting ligand or

antibody. PEG can meet the requirements above for multifunction of MNPs by coating

them (Yallapu, Foy, Jain, & Labhasetwar, 2010).

15

1.6.5 Folic Acid Directed Drug Delivery

Folic acid is a form of vitamin B9. Folic acid is important to synthesize nucleic acids

(DNA and RNA). Folic acid is also vital for rapid cell division and growth and for

production of healthy red blood cells. Consuming enough folic acid during pregnancy

could prevent major birth defects (Medical News Today, 2013). The structure of folic

acid is given in Figure 1.6.

Figure 1.6: Structure of folic acid (Sigma-Aldrich, n.d.-a).

Folic acid targets to the folate receptor which is a cell surface receptor. This folate

receptor is overexpressed by many different cancer cells while healthy cells have low

expression of folate receptor. Such tumors with overexpressed folate receptors are

epithelial, ovarian, cervical, breast, lung, kidney, colorectal, and brain tumors. Folic

acid is a non-immunogenic and inexpensive molecule and also it can retain its stability

16

over large scale of temperatures and wide range of pH values. Conjugation of folic acid

to drug carrier does not affect the ability of folic acid to interact with the folate receptor.

Binding of folic acid to its receptor causes endocytic internalization of the carrier

(Figure 1.7). Drop of the endosomal pH to five triggers dissociation of the folate from

its receptor and then, causes release of the drug (Zwicke, Mansoori, & Jeffery, 2012).

As a result, these properties of folic acid make it very useful targeting molecule for

application of targeted drug delivery system in cancer.

Figure 1.7: Representative picture of the folate receptor-mediated endocytosis

pathway (Ashley et. al, 2011).

1.7 Synthesis Methods of Magnetic Nanoparticles

There are many effective synthesis methods for production of MNPs with desired size,

high stability, and unique dispersion. Co-precipitation, thermal decomposition,

microemulsion and hydrothermal synthesis techniques are used to synthesize high-

17

quality magnetic nanoparticles. These four methods have advantages and drawbacks

(Table 1.1). The simplest and more preferred method is co-precipitation technique to

synthesize MNPs. Thermal decomposition method provides control on size and

morphology of the nanoparticles. Monodispersed MNPs with diverse morphologies can

also be synthesized via microemulsion. Disadvantage of this method is consumption of

solvent in excess amount. Hydrothermal synthesis method is an infrequently used

method to produce magnetic nanoparticles; however, magnetic nanoparticles with high-

quality are synthesized via this method. Until now, most studies are conducted on

magnetic nanoparticles that have been synthesized by using thermal decomposition and

co-precipitation. By these two methods, they are produced in large amounts (Lu,

Salabas, & Schüth, 2007).

Table 1.1: Comperassion of MNP Synthesis Methods (Lu et al., 2007)

Synthetic

Method

Synthesis Reaction

Temp.

(oC)

Reaction

Period

Solvent Surface-

Capping

Agent

Size

Distribution

Shape

Control

Yield

Co-

precipitation

Very

simple,ambient

conditions

20-90 Minutes Water Needed,

added

during or after

reaction

Relatively

narrow

Not

good

High/scalable

Thermal

Decomposition

Complicated, inert

atmosphere

100-320 Hours-days

Organic compound

Needed, added

during

reaction

Very narrow Very good

High/scalable

Microemulsion Complicated,

ambient

conditions

20-50 Hours Organic

compound

Needed,

added

during reaction

Relatively

narrow

Good Low

Hydrothermal

Synthesis

Simple, high

pressure

220 Hours

ca.day

Water-

ethanol

Needed,

added

during reaction

Very narrow Very

good

medium

http://onlinelibrary.wiley.com/doi/10.1002/anie.200602866/full#tbl3

18

1.8 Objective of the Study

The objectives of this study can be summarized as follows:

To synthesize Fe3O4 magnetic nanoparticles by thermal decomposition.

To cover MNP with polyethylene glycol (PEG) monooleate

To conjugate folic acid to PEG modified MNP.

To characterize the synthesized MNPs, PEG coated MNP and FA conjugated

PEG coated MNP.

To investigate doxorubicin-loading on folic acid conjugated PEG coated MNPs

and doxorubicin release from them.

To visualize the internalization of magnetic nanoparticles by HeLa cells.

To investigate cytotoxicities of FA conjugated PEG coated MNPs and

Doxorubicin loaded form of them to sensitive and drug resistant lines of HeLa

cervical cancer cells.

19

CHAPTER 2

2 MATERIALS AND METHODS

2.1 Materials for Magnetic Nanoparticle Synthesis

Iron(III) acetylacetonate (Fe(acac)3), oleic acid, oleylamine, polyethylene monooleate

(PEG), folic acid, dicyclohexyl carbodiimide (DCC) and dimethylsulfoxide (DMSO)

were purchased from Sigma-Aldrich (U.S.A.). Nitrogen gas was obtained from Asya

Gaz (Turkey).

2.2 Synthesis of Magnetic Nanoparticles

In literature, there are many methods including co-precipitation, thermal decomposition

and reduction, micelle synthesis, hydrothermal synthesis, and laser pyrolysis techniques

synthesize high-quality magnetic nanoparticles (Lu et. al, 2007).

In this research, thermal decomposition method was used to synthesize Fe3O4 magnetic

nanoparticles. Generally, the thermal decomposition is decomposition of iron precursor

in a high-boiling temperature stabilizing surfactant solvents such as trioctylamine, oleic

acid, oleylamine and so on (Zhao, Zhang, & Feng, 2012). However, the synthesized

magnetic nanoparticles have hydrophobic surfactant on their surface, so the magnetic

nanoparticles are soluble in hydrophobic solution such as hexane and ethanol.

In this study, Fe3O4 magnetic nanoparticles were prepared by the thermal

decomposition of Fe(acac)3 in oleic acid and oleylamine solution at 3000C (Maity,

20

Choo, Yi, Ding, & Xue, 2009). Fe(acac)3 was dissolved in the mixture of oleic acid and

oleylamine, and then mechanically stirred under a flow of nitrogen. The solution was

dehydrated at 1200C for 1h, and then quickly heated to 300

0C and kept at this

temperature for 1h. Heat source was removed to cool the black solution. Ethanol was

added to the black solution and magnetic nanoparticles were precipitated by magnet.

The nanoparticles were washed several times with ethanol. The synthesized Fe3O4

magnetic nanoparticles (OL-Fe3O4) which have oleic acid and oleylamine on their

surface were labeled as MNPs.

2.3 Synthesis of PEG Coated Magnetic Nanoparticles

MNP has hydrophobic property due to the hydrophobic surfactants (oleic acid and

oleylamine) on the surface of Fe3O4 particles. To be biocompatible, soluble in

hydrophobic environment and able to carry drug, the magnetic nanoparticles should be

coated with an appropriate polymer or dendrimer.

In this research, OL-Fe3O4 magnetic nanoparticles were mixed with PEG in distilled

water and the solution was stirred overnight at room temperature. Then, PEG coated

MNP (PEG-MNP) was precipitated by magnet. The nanoparticles were washed with

distilled water until clear supernatant was observed.

2.4 Folic Acid Modification of PEG-MNP

PEG-MNP was modifed with activated folic acid (Zhang, Rana, Srivastava, & Misra,

2008). Folic acid (FA) is dissolved in dimethyl sulfoxide (DMSO). Dicyclohexyl

carbodiimide (DCC) is added to the solution and the solution is stirred for 2 h in a

21

nitrogen atmosphere. Then, PEG-MNP is added and continuously stirred for 2 h in a

nitrogen atmosphere. Finally, FA modified PEG-MNP (FA-MNP) was washed twice

with distilled water and stored in distilled water at room temperature.

2.5 Drug Loading

In this study, the absorptions of the different concentrations of doxorubicin (Dox) were

measured to determine the standard curve of its absorption versus concentration.

Standard curve were used to measure concentration of Dox remaining in solution after

Dox loading to FA-MNP.

Different concentrations of Dox were added to the eppendorf tubes containing same

amount of FA-MNP. The tubes were placed to the rotary shaker (Biosan Multi RS-60

Rotator). Then, the tubes were shaked at room temperature for 24 hours. Dox loaded

FA-MNP was precipitated by application of magnet. Then, supernatant was collected

and absorption of supernatant was measured to determine the remained Dox

concentration.

2.6 Drug Release

After drug loading process, drug-loaded FA-MNPs in eppendorf tubes are washed with

distilled water and same amount of acetate buffers with pH 4.13 and 5.14, and pH 7.4

added to the tube. Then, it is placed to rotator shaker. After 3 hours, particles are

precipitated by magnet and supernatant is taken to new tube for measurement of drug

absorbance. After that, the solvents were added to the tube and the tubes were rotated.

This process was repeated several times for 72 h. Finally, concentration of released drug

22

is calculated from its absorbance at 480 nm measured by a UV spectrophotometer

(Multiskan GO, Thermo Scientific) via the slope of standard curve.

2.7 Cell Culture

2.7.1 Cell Line and Culture Condition

Parental sensitive HeLa and doxorubicin resistant HeLa (HeLa/Dox) cells were

cultured in RPMI 1640 medium (Thermo Scientific, USA) with 10% heat inactivated

fetal bovine serum (FBS) (Biochrome, Germany) and 0.2% gentamycin (Biological

Industries, Israil). Incubation condition of cells in a Heraeus incubator (Hanau,

Germany) involved 37oC, a humidified atmosphere with 5% CO2.

2.7.2 Subculturing

Subculturing is applied to cells that have 80% cell confuency in a flask to prevent them

from dying before all nutrients in the medium are consumed. Firstly, old medium is

taken from the flask and cells are washed with 5 ml phosphate buffered saline (PBS) for

T-75 filter capped tissue culture flask. It is important that washing the cells with PBS

prevents trypsin from inhibiton caused by medium and waste products. Secondly, 1 mL

trypsiın was added to the flask and the flask is put into the incubator with 37oC, 5%

CO2, humidified condition for a few minutes. During incubation, trypsin detachs cells

from the surface of the flask via digestive enzymatic activity. Thirdly, 5 ml medium

supplemented with serum is added to flask for inhibition of trypsin and used to

homogenize cells by pipetting. Cell suspension is transferred to 15 mL Falcon tube

(Greiner) and centrifuged at 1000 rpm for 5 minutes. Supernatant is discarded and 3 mL

fresh medium is added to tube to resuspend the pellet. Then, a proper amount of cell

23

suspension is transferred to the flask and total volume is completed to 12 mL by

medium addition. For HeLa/Dox cells, there is one additional step in which 12 µL of

500 µM doxorubicin solution is added to their medium to maintain resistance. Finally,

cells are incubated at 37oC.

2.7.3 Cell Freezing

In a tissue culture flask, when cells reach at least 80% confluence or more, they are

trypsinated and detached. The detached cells are resuspended in 4 mL of medium and

centrifuged at 1000 rpm for 5 min. Supernatant is discarded and cells are resuspended

with 4 ml PBS. Again, the cells are centrifuged at 1000 rpm for 5 min. Supernatant is

removed and cells are suspended in 1 mL of cold freezing medium (90% (v/v) complete

growth medium supplemented with 10% (v/v) DMSO (Sigma-Aldrich, USA)) to have a

final concentration of approximately 2x106 cells per ml. This cell suspension is

transferred into the cryovials (Greiner) and maintained at 4ºC for 30 min, then kept in -

20ºC for 3-4 hours before the overnight incubation at -80ºC. Finally cryovials are

transferred to liquid nitrogen container for long term storage.

2.7.4 Cell Thawing

The cryovials containing the frozen cells are taken from liquid nitrogen container and

immediately placed into a 37°C water bath. Quickly after cells are thawed, they are

transferred to a falcon tube and 4 mL pre-warmed growth medium is added on the

thawed cells. Then, cell suspension is centrifuged at 1000 rpm for 5 min. Supernatant is

discarded. The cells are gently resuspended in growth medium, and transferred into a

cell culture flask. Finally, cell containing flask is incubated at 37oC.

24

2.7.5 Cell Counting

Trypan blue and a hemocytometer as a microscope slide are used for viable cell

counting. Trypan blue is applied to distinguish viable cells from dead cells. This means

that trypan blue dye selectively penetrates membranes of the dead cells and colors them

blue while cell membranes exclude viable cells from trypan blue staining and alive cells

are not stained. In this method, 225 μL of the cell suspension to be counted is mixed

with 25 μL trypan blue in an eppendorf tube. The shoulders of the hemocytometer are

moisturized and the coverslip is affixed on them. Some cell suspension containing

trypan blue is loaded to the chamber of hemocytometer at the edge of the cover slide.

The grid lines of the haemocytometer are focused under light microscope. The number

of cells in the area of 16 squares which is composed of 256 small squares is counted.

During cell counting, live cells (unstained with trypan blue) are always counted and

stained cells are ignored. Cell suspension to be used for an experiment must have more

than 90 % live cells of total cell number. Otherwise, cell suspension cannot be used for

any experiment. Cell number in 1 mL cell suspension is calculated by following

formula:

Cell number mL

2.8 Internalization of Nanoparticles

Internalization of nanoparticles by HeLa cells was investigated by Purssian blue

staining (Sigma-Aldrich) and/or using fluorescent property of doxorubicin loaded to

FA-MNP.

http://en.wikipedia.org/wiki/Microscope_slide

25

2.8.1 Detection of Internalized Nanoparticles by Purssian Blue Staining

Iron oxide nanoparticles was stained by purssian blue staining method (Boutry et. al,

2009). The nanoparticles that were internalized by HeLa cells were detected by this

technique. This method provides two solutions, working iron solution and working

pararosaline solution. To prepare working iron stain solution, potassium ferrocynanide

solution and HCl (1:1 v/v) are mixed in a falcon tube. For preparation of working

pararosaline solution, pararosaline solution is added to dH2O to be 2% (v/v) in a falcon

tube. Acid ferrocyanide in working iron stain solution reacts with iron causing

production of blue color or dark blue in case of heavy deposit of iron, while working

pararosaline solution stains cell producing red color in nucleus and pink color in

cytoplasm.

Procedure of purssian blue staining is modified. Firstly, cover slides were placed inside

the wells of 6-well plate. 200000 cells were seeded to each well. Old medium was

removed after 24 hours and cells were washed with PBS solution. 2mL fresh media

containing different concentration of Fe3O4 particles were added to wells. Cells were

incubated at 37oC and 5% CO2 for 7 hours. Then, cells were washed 5 times with PBS

to get rid of MNPs that has not been internalized by cells. Working pararosaline

solution is added on cells in wells and cells are incubated for 10 minutes. The cells are

washed one time with distilled water. Working pararosaline solution is added on cells in

the flask and the flask is incubated for 5 minutes. The cells are washed one time with

distilled water and left to dry. Finally, the cells are observed under light microscopy and

photographed.

26

2.8.2 Detection of Internalized Nanoparticles by Fluorescent Microscopy

Doxorubicin is a chemotherapy drug and has a fluorescent property. When it is loaded

to FA-MNPs, they gain fluorescent property and can be detected by fluorescent

microscopy.

Firstly, 200000 cells were seeded to each well of 6-well plate. Old medium was

removed after 24 hours and cells were washed with PBS solution. Then, 2mL fresh

media containing different concentrations of Dox loaded MNPs were added to wells.

Cells were incubated at 37oC and 5% CO2 for 7 hours. Then, cells were washed 5 times

with PBS to get rid of the magnetic nanoparticles that have not been internalized by

cells. The cells are observed under fluorescent microscope and photographed.

2.8.3 Cell Proliferation Assay

XTT cell proliferation assay is a colorimetric assay for quantification of cell

proliferation, viability, and cytotoxicity of a drug. In this assay, XTT reagent (a

tetrazolium salt) is reduced to formazan by the mitochondrial enzymes in live cells.

50 μL cell suspension were added to the wells of 96 well plate (Figure 2.1) and

incubated at 37oC and 5% CO2 humidified atmosphere for 24 h. After the incubation,

medium is discarded and cells were washed with PBS.

27

Figure 2.1: Representative picture of cell seeded 98 well plate. Gray wells

represent cell seeded ones.

Then, 200 μL of highest drug dose (HDD) containing medium (10 μM doxorubicin for

HeLa or 1000 μg/mL nanoparticles for HeLa and HeLa/Dox) is added to each wells of

3rd

column and 50 μL fresh medium is added to remaining wells. 150 μL of HDD

containing medium were transferred from 3rd

column to 4th

column and mixed well with

50 μL medium in each wells of 4th

column. 150 μL of drug containing medium were

transferred from 4th

column to 5th

column and mixed well with 50 μL medium in each

wells of 5th

column. This dilution was continued till 12th

column (Figure 2.2). 50 μL

medium was added to all wells of the plate, so HDD became half of the strating

concentration (5 μM doxorubicin for HeLa or 500 μg/mL nanoparticles for HeLa and

HeLa/Dox). The plate was incubated at 37oC and 5% CO2 humidified atmosphere for 72

h. After that, 50 μL activated XTT is added to each wells and incubated for 4 h.

Absorption of samples in wells was measured at 492 nm. 1st column was medium

28

control and 2nd

column was cell control. To find out the absorbance of only cells,

absorbance of 1st column was subtracted from absorbance of 2

nd column. This

absorbance of cells was accepted as 100% proliferation. Absorbance of drug treated

cells in column 3-12 were calculated by subtracting average absorbance of A and H

wells from that of cell seeded wells in same column. Percentage of cell proliferation

was calculated by dividing absorbance of column to that of control.

Figure 2.2: Representative picture of drug dilution in 96 well plate.

2.9 Statistical analysis

Results gathered from at least two independent experiments are expressed as mean

SEM and were analyzed using GraphPad Prism Version 5.01 (GraphPad Inc., USA)

29

with one-way ANOVA followed by Tukey’s Multiple Comparison Test. Resullts were

significant when p<0.05.

31

CHAPTER 3

3 RESULTS AND DISCUSSION

3.1 Chemical Characterization

Oleic acid and oleylamine on their surfaces make the synthesized MNPs hydrophobic

beause they are produced by decomposition of Fe(acac)3 in mixture of these two

surfactants in high temperature. Then, the synthesized magnetic nanoparticles are

coated with PEGmonooleate by hydrofobic interaction between hydrofobic surfactants

on MNPs and oleate part of PEG monooleate in distilled water which is hydrophilic

solvent.

3.1.1 Chemical Characterization of MNPs

Magnetic nanoparticles are synthesized in small size and narrow size range via termal

decomposition method. The sizes of magnetic core and morphological properties were

observed through TEM images. The chemical groups and chemical interactions

involved in synthesized MNPs were identified using the FTIR methods. Crystal

structures of synthesized MNPs were analyzed by XRD. The qualitative and

quantitative information about the volatile compounds of the nanoparticles have been

provided by TGA. Magnetic properties of MNPs were determined through VSM

analyses.

32

3.1.1.1 Transmission Electron Microscopy of MNPs

Size and morphology of synthesized OL-Fe3O4 magnetic nanoparticles (MNPs) have

been observed by Transmission Electron Microscopy (TEM). Obtained images (Figure

3.1) showed that MNPs are almost spherical and have more uniform size distribution.

The average diameter of MNPs is approximately 10 nm. Images of MNPs in two

solvents (ethanol and hexane) are taken by TEM. Ethanol can dissolve both

hydrophobic and hydrophilic substances because it is a versatile solvent while hexane

dissolves only hydrophobic materials due to its hydrophobic property. As a result,

MNPs are more dispersed in hexane than in ethanol because MNPs are hydrophobic due

to oleic acid and oleylamine on their surface.

33

a)

b)

Figure 3.1: TEM images of MNPs (a): in Ethanol, b): in Hexane)

34

3.1.1.2 X-Ray Diffraction

The crystal structure of sythesized iron oxide (OL-Fe3O4) nanoparticles was determined

by XRD. Diffraction peaks at 30.25, 35.41, 43.41, 57.30, 62.70 and 74.49 2θ (degrees)

which are corresponding to specific diffractive plane indexes (220), (311), (400), (422),

(511), (440) and (533) respectively (Figure 3.2). XRD patterns of OL-Fe3O4 were

examined by comparing to peaks of standard magnetite in JCPDS file (PDF no: 01-075-

1609). All peaks are characteristic peaks of the magnetite (Fe3O4) crystals that have an

inverse cubic spinel structure. XRD results revealed the presence of the Fe3O4 crystals

in the synthesized nanoparticles OL-Fe3O4. The peaks shown in the XRD pattern of the

prepared sample are sharp and intense, indicating crystallinity of the sample.

Figure 3.2: X-Ray powder diffraction (XRD) patterns of synthesized iron oxide

(OL-Fe3O4) nanoparticles.

35

3.1.1.3 Fourier Transform Infrared Spectroscopy of MNPs

In order to confirm the chemical composition of synthesized nanoparticles, fourier

transform infrared spectroscopy analysis were obtained. The peak located in the 583 cm-

1 region, characteristic for the Fe-O group MNPs’ spectra, confirms that the products

contain magnetite (Fe3O4). All characteristic peaks of OL-Fe3O4 were present in Figure

3.3. The band at 580 cm-1

corresponded to the vibration of the Fe–O bonds in the

crystalline lattice of Fe3O4 (Li, Wei, Gao, & Lei, 2005). The bands at 2852 and

2922 cm-1

were attributed to the asymmetric CH2 methylene stretch and the symmetric

CH2 stretch in oleic acid, respectively (Maity et. al, 2009). The band at 1409 cm-

1 corresponded to the CH3 umbrella mode of oleic acid. The bands at 1427 and

1523 cm-1

were attributed to the asymmetric (COO) carboxyl and symmetric (COO)

stretch vibration band (Yang, Peng, Wen, & Li, 2010).

Figure 3.3: FTIR spectrum of MNPs.

0

20

40

60

80

100

120

05001000150020002500300035004000

Tran

smet

ance

(%

)

Wavelength (cm-1)

2921

1409

2852

1427

1523

583

36

3.1.1.4 TGA (Thermal Gravimetric Analysis) of MNPs

The percents of Fe3O4 and OL (Oleic acid and Oleylamine) in the nanoparticles were

measured by thermo-gravimetric analyzer. The TGA analysis of MNPs (OL-Fe3O4)

provides qualitative and quantitative information about the volatile components. The

TGA analysis of MNPs has two weight loss phases (Figure 3.4). The first weight loss

(~2%) belongs to the decomposition of ethanol absorbed by OL molecules on the

surface of Fe3O4 particles. In the second phase of weight loss which is ~10% of total

mass, OL molecules were decomposed by increasing the temperature. TGA result also

suggests that MNPs have OL molecules on their surface.

Figure 3.4: Thermal gravimetric analyses of MNPs.

86

88

90

92

94

96

98

100

102

0 200 400 600 800 1000

Wei

ght

(%)

Temperature (oC)

10%

~ 2%

37

3.1.1.5 VSM (Vibrating Sample Magnetometer)

Magnetic hysteresis curve was obtained by vibrating sample magnetometer. The applied

magnetic field was changed and magnetization properties of synthesized OL-Fe3O4

nanoparticles were measured at 37˚C. Remanence (magnetization of particles after

removal of magnetic field) and coercivity (a measurement of a reverse field needed to

drive the magnetization to zero after saturation) were not observed in the hysteresis

curve. This phenomenon proved that all nanoparticles synthesized in this study are

superparamagnetic. The saturated magnetization (MS) of MNPs is 55 emu/g (Figure

3.5).

Figure 3.5: Vibrating sample magnetometer (VSM) results show magnetization

and demagnetization curves of MNPs (55 emu/g) at 37˚C.

-80

-60

-40

-20

0

20

40

60

80

-30000 -20000 -10000 0 10000 20000 30000

Mo

me

nt/

Mas

s (e

mu

/g)

Field/G

1 (Demagnetization) 1 (Magnetization)

38

3.1.2 Chemical Characterization of PEG-MNP

The synthesized magnetic nanoparticles are coated with PEG monooleate by hydrofobic

interaction between hydrofobic surfactants on MNPs and oleate part of PEG

monooleate in distilled water which is hydrophilic solvent.

Sizes and morphologies of PEG-MNPs were analyzed by Transmission Electron

Microscopy (TEM) images. The chemical groups and chemical interactions involved in

PEG-MNPs were analyzed by using the FTIR methods. The qualitative and quantitative

information of nanoparticles have been provided by TGA.

3.1.2.1 TEM Characterization of PEG-MNP

Size and morphology of PEG-MNPs were examined by TEM. TEM results (Figure 3.6)

demonstrated that PEG-MNPs had almost spherical and more uniform size distribution.

The average diameter of MNPs is approximately 15 nm. Images of PEG-MNPs are

taken by TEM.

39

a)

b)

Figure 3.6: TEM images of PEG- MNPs.

40

3.1.2.2 Fourier Transform Infrared Spectroscopy (FTIR) of PEG-MNP

The FTIR analyses of PEG-MNP were performed to confirm that MNPs were coated

with PEGmonooleate. The peaks at 946 cm-1

and 1109 cm-1

indicated the stretching

vibration of functional CH2 group and the stretching vibration of functional group of C–

O of PEG respectively while the absorption band observed at 1735 cm-1

was caused by

C=O carbonyl group of PEG (Figure 3.7). Thus, these results confirmed that MNPs

were successfully coated with PEG.

Figure 3.7: FTIR spectrum of PEG-MNPs.

Oleate group of PEG monooleate formed hydrophobic interaction with oleic acid on the

surface of MNPs. Thus, this interaction formed interpenetration layer and PEG formed

secondary layer by coating MNPs (Figure 3.8). The intense peak at 1705 cm-1

was due

to the C=O stretching of oleate part of PEG monooleate. This peak was showed at 1710

300800130018002300280033003800Wavelength (cm-1)

PEG-MNPMNP

1705 1109 945 1735

41

cm-1

for second layer of oleic acid on MNPs by Yan et. al (Yang et. al, 2010). The FTIR

result showed that MNPs were successfully coated with PEG.

Figure 3.8: Schematic representation of PEG coated MNPs (Yang et. al, 2010).

3.1.2.3 Thermal Gravimetric Analysis (TGA)

TGA was used to show existence of PEG on the surface of particles and to determine

amount of PEG by decomposition of particles. TGA analysis revealed that MNPs had

12% weight loss due to decomposition of oleic acid and oleylamine while PEG-MNPs

had 44% weight loss (Figure 3.9). 32% difference showed that PEG exists on the

surface of the nanoparticles.

42

Figure 3.9: Thermal gravimetric analyses of PEG- MNPs.

40

50

60

70

80

90

100

110

0 100 200 300 400 500 600 700 800

We

igh

t (%

)

Temperature (°C )

MNP

PEG-MNP

12%

32%

43

3.1.3 Chemical Characterization of FA-MNPs

The FTIR analysis was made to demonstrate FA modification of PEG-MNPs.

3.1.3.1 Fourier Transform Infrared Spectroscopy of FA-MNPs

The FTIR result of FA-MNP showed that the characteristic absorption peaks 1433 and

1606 cm-1

of FA-MNP (Figure 3.10) were also shared by the FTIR spectrum of FA

(Appendix B.2). The characteristic band of 1433 cm-1

wavelength was belonging to the

phenyl ring of folic acid. 1606 cm-1

band indicated –NH2 bending vibration of FA

(Keskin, 2012). Because of these bands, modification of PEG-MNPs with FA was

successfully achieved.

Figure 3.10: FTIR spectrum of FA-MNPs.

300800130018002300280033003800

FA-MNP

PEG-MNP

1606 1433

44

3.2 Drug Loading

Different concentrations of doxorubicin were mixed with 500 μg/ml FA-MNPs in Tris

buffer having pH 7 by rotary shaker at room temperature for 24 h. Increase in total drug

concentration in mixture decreased the percentage of the doxorubicin loaded to FA-

MNPs (Figure 3.11). This did not show the concentration of drug loaded to FA-MNPs.

Figure 3.11: Percentages of doxorubicin loaded to FA-MNPs in different

concentrations of doxorubicin.

In 1724 μM doxorubicin concentration, ~ 29% of total doxorubicin was loaded to FA-

MNPs. This was seen that ~29 % drug loading could be considered as low drug loading

capacity of MNPs. However, ~29 % drug loading was equal to around 505 μM

doxorubicin loaded to FA-MNPs which was the highest drug concentration in molarity

(Table 3.1). Loaded drug concentration of drug was calculated from the absorption by

0

10

20

30

40

50

60

70

80

90

0 500 1000 1500 2000

Load

ed

Dru

g (%

)

Total Drug Conc (μM)

45

using standard curve of doxorubicin (Appendix D). However, 78 µg of doxorubicin per

mg of nanocomposite was sufficient to kill cancer cells (Chen et. al, 2011). In this

study, 290 µg of doxorubicin was successfully loaded to 1 mg nanoparticles. FA-MNPs

had nearly 50% doxorubicin loading capacity (Zhang et al., 2008), which was higher

than that was found in this study. There are some factors that affect the loading capacity

of particles. First of all, size of PEG could have role on loading capacity. If the polymer

that coats the particles is longer, the particles could have higher drug loading capacity.

Also, pH of environment could affect the amount of drug loaded to the particles.

Moreover, buffer type used during drug loading could increase or decrease drug

loading. Finally, particles used in this study were in dH2O; in other words, particles

were moisture. When dried particles were used for drug loading, more drugs could be

loaded as moisture enters between polymers on particles. However, they cannot be as

dispersed in a solvent after the particles were dried. This is the reason why the particles

were not dried.

Table 3.1: Concentration of doxorubicin loaded to FA-MNPs.

At pH 7

Total Drug

Conc (μM)

Loaded Drug Conc

± SEM (μM) 1724 505.01 ± 11.23

862 273.40 ± 5.01

431 154.70 ± 5.06

215.50 89.63 ± 5.24

107.75 54.02 ± 2.65

53.88 32.76 ± 2.17

26.94 22.25 ± 0.71

javascript:popupOBO('CL:0000000','C1JM11195G')

46

3.3 Drug Release

The release profiles of doxorubicin from Dox-FA-MNPs in acetate buffer with pH 4.13

and pH 5.14 and pH 7.4 at 37oC were measured at 480 nm in 3 h intervals by UV

spectrophotometer. Drug release was investigated at acidic pH to mimic the endosomal

condition. Moreover, the release temperature was 37oC which is equal to body

temperature and also incubation temperature of cells. According to the results, at low

pH 4.13, drug release from nanoparticles occurred faster and higher than at two other

pH values (Figure 3.12). Acidity of environment affected the release of doxorubicin in

vitro and the more the acidic condition, the more release rate of doxorubicin when

compared two different pH values. Percentages of drug releases from Dox-FA-MNPs

were 15.74 %, 14.27 % and 10.01 % in acetate buffers with pH 4.13, pH 5.14 and pH

7.4 respectively in 72 h.

Figure 3.12: Released doxorubicin concentration from Dox-FA-MNPs in acetate

buffers with different pH values.

0

10

20

30

40

50

60

70

80

90

0 20 40 60 80

Cu

mu

lati

ve D

rug

Re

leas

e (

μM

)

Time(Hour)

pH 4.13

pH 5.14

pH 7.4

47

Table 3.2: Concentration of released doxorubicin from Dox-FA-MNPs in different

solution and different pH Values.

3.4 Cell Culture Studies

3.4.1 Development of Drug Resistant Cell Line

Doxorubicin resistant (HeLa/Dox) cell line was developed from parental (HeLa) cell

line in our laboratory by gradually increasing drug concentration of medium in which

cells grow and proliferate. Starting from the low drug concentration, drug concentration

in medium was increased after cells were not affected by certain drug dosage. Finally,

HeLa cells became resistant to 500 nM doxorubicin. For the confirmation of resistance,

the half maximal inhibitory concentration (IC50) values of doxorubicin, which were

determined by XTT cell proliferation assay, for HeLa and HeLa/Dox were compared.

IC50 value of doxorubicin for parental HeLa cells was 2.725 ± 0.147 μM (Figure 3.13)

while IC50 value of doxorubicin for HeLa/Dox cells is 100.0 ± 4.5 μM (Figure 3.14).

pH 4.13 pH 5.14 pH 7.4

Time (h)

Drug Conc ± SEM (μM)

Drug Conc ± SEM (μM) Drug Conc ± SEM (μM)

0 0.00 0.00 0.00 0.00 0.00

3 48.09 ± 0.68 23.51 ± 0.28 5.83 ± 0.05

6 56.28 ± 0.74 32.31 ± 0.39 11.70 ± 0.61

9 58.99 ± 0.73 39.02 ± 0.47 16.21 ± 0.86

12 61.21 ± 0.78 44.60 ± 0.53 20.80 ± 1.09

15 63.07 ± 0.73 48.45 ± 0.54 24.67 ± 1.11

18 65.72 ± 0.87 52.58 ± 0.55 29.00 ± 1.34

21 68.08 ± 1.03 56.17 ± 0.42 33.28 ± 1.66

24 69.97 ± 0.99 59.03 ± 0.43 38.03 ± 1.81

38 72.64 ± 1.10 62.95 ± 0.33 43.06 ± 1.81

48 74.54 ± 1.08 66.68 ± 0.22 46.37 ± 1.91

72 77.58 ± 1.17 70.74 ± 0.31 50.36 ± 1.06

48

These results showed that HeLa/Dox cells were 37 times resistant to doxorubicin

compared to HeLa cells.

0.38

0.50

0.67

0.89

1.19

1.58

2.11

2.81

3.75

5.00

0

20

40

60

80

100

Doxorubicin Concenration (M)

%C

ell P

rolife

rati

on

Figure 3.13: Cell proliferation profile of HeLa cells treated with increase in

Doxorubicin concentration

49

11 15 20 27 36 47 63 84 113

150

0

20

40

60

80

100

Doxorubicin Concenration (M)

%C

ell P

rolife

rati

on

Figure 3.14: Cell proliferation profile of HeLa/Dox cells treated with increase in

Doxorubicin concentration

3.4.1.1 Internalization of Nanoparticles

FA-MNPs internalized by HeLa cells were investigated by Purssian blue staining and

Dox-FA-MNPs up taken by HeLa were detected by using fluorescent property of

doxorubicin under fluorescent microscope.

3.4.1.2 Detection of FA-MNPs in HeLa Cells by Purssian Blue Staining

Purssian blue staining method stains cellular components and iron core of particles in

different colors. By this technique, cell and cell components are stained purple while

nanoparticles are blue or dark blue in order to observe internalization of the

nanoparticles. Different concentrations of FA-MNPs were given to HeLa cells and

50

incubated at 37oC for 7h. Then, the cells were stained by purssian blue staining method.

Increasing concentration of the nanoparticles increased the amount of the particles in

the cells (Figure 3.15). Non-treated HeLa cells were seen in only pink color while FA-

MNPs-treated HeLa cells were colored as both pink and blue. This result showed that

FA-MNPs were internalized by the cells. Also, internalized nanoparticles in low dose

were localized together in cells while in highest dosage; they covered all around the

cytoplasm. The internalization of nanoparticles by the cells is started as an interaction

(adsorbing) followed by vesicle formation and then the formed nanoparticle-containing

vesicles are internalized by endocytosis (Osaka. Nakanishi. Shanmugam. Takahama. &

Zhang. 2009). The nanoparticles generally were located near nucleus. As a result, it was

seen that the particles were endosomally internalized by HeLa cells.

a) b)

Figure 3.15: Images of HeLa cells treated with 0 μM (a), 25 μg (b), 50 μg (c), 100

μg (d), 150 μg (e) and 200 μg FA-MNPs (f).

51

c) d)

e) f)

Figure 3.16: Images of HeLa cells treated with 0 μM (a), 25 μg (b), 50 μg (c), 100

μg (d), 150 μg (e) and 200 μg FA-MNPs (f) (Continued).

52

3.4.1.3 Detection of Dox-FA-MNPs in HeLa Cells by Fluorescent Microscopy

Other detection method for internalized nanoparticles was using the fluorescent

property of doxorubicin. As doxorubicin was loaded to FA-MNPs. they gained its

fluorescent property. HeLa cells were treated with doxorubicin and different

concentration of Dox-FA-MNPs and incubated at 37oC for 7 h. In order to prevent the

background fluorescence from non-internalized Dox-FA-MNPs. HeLa cells were

extensively washed with PBS. Only doxorubicin treated HeLa cells had fluorescence in

their nucleus because doxorubicin targets nucleus while due to endosomal

internalization of the particles, there was intense fluorescent emission in cytoplasm of

Dox-MNP treated HeLa cells (Figure 3.16).

53

a) b)

c) d)

Figure 3.17: Images of HeLa cells treated with doxorubicin (a), 50 μg/mL (b), 150

μg/mL (c) and 200 μg/mL (d) Dox-FA-MNPs.

54

3.4.2 Antiproliferative Activity of Nanoparticles

The antiproliferative effects of drug loaded MNPs (Dox-MNP) and non-drug loaded

MNPs (FA-MNPs) were determined by XTT cell proliferation assay. For this, HeLa

and HeLa/Dox cells were treated with different doses (38-500 μg/mL) of FA-MNPs and

Dox-FA-MNPs and incubated for 72 h.

The result demonstrated FA-MNPs did not have cytotoxity on HeLa cells while those of

Dox-FA-MNPs caused significant decreases on cell proliferation of HeLa cells (Figure

3.17). As a result, FA-MNPs were biocompatible nanoparticles and could be used as

drug carriers due to antiproliferative effects of their drug loaded forms.

38 50 67 89 119

158

211

281

375

500

0

50

100

150FA-MNP

Dox-FA-MNP

***

***

***

***

********

*

***

***

***

Particle Concentration (g/mL)

% C

ell P

rolife

rati

on

Figure 3.18: Antiproliferative effects of FA-MNPs and Dox-FA-MNPs on HeLa

cell line. Results were obtained from three independent experiments, represented

as mean SEM (*** Results were significant with a p<0.05).

55

Antiproliferative effect of Dox-FA-MNPs on HeLa/Dox cells was investigated by XTT

cell proliferation assay in order to see whether particles overcome the drug resistance of

the HeLa/Dox cells. XTT dependent cell proliferation assay result showed that Dox-FA-

MNPs did not have a significant cytotoxicity on HeLa/Dox cells (Figure 3.18). This

could be due to overexpression of P-glycoprotein (cell surface efflux transporter) by

doxorubicin in HeLa because doxorubicin upregulates P-gylcoprotein in doxorubicin

resistant MCF-7 cells which is main mechanism of multi drug resistance (Dönmez &

Gündüz, 2011). Drug release from internalized Dox-FA-MNPs could be slow, so the

inner drug concentration could not reach a critical level to kill cells and already released

drug in cells could also be pumped outside by transpoter proteins. Therefore, Dox-FA-

MNPs could not show cytotoxic effects on HeLa/Dox cells.

Figure 3.19: Antiproliferative effects of FA-MNPs and Dox-FA-MNPs on

HeLa/Dox cell line. Results were obtained from three independent experiments,

represented as mean SEM (*** Results were significant with a p<0.05).

38 50 67 89 119

158

211

281

375

500

0

50

100

150

FA-MNP

Dox-FA-MNP**

*

***

***


% C

ell P

rolife

rati

on

57

CHAPTER 4

4 CONCLUSION

1. Synthesized MNPs had approximately 10 nm size and a spherical shape. The

core of MNPs contained magnetite (Fe3O4). MNPs were also superparamagnetic

iron oxide nanoparticles. They had oleic acid and oleylamine on their surface,

which made them hydrophobic.

2. MNPs were coated with PEG via hydrophobic interaction between hydrophobic

surface of MNPs and hydrophobic part (oleate) of PEG monooleate. FTIR

showed MNPs coated with PEG. Size of PEG-MNPs was smaller than 20 nm.

Due to PEG coating. PEG-MNPs were bigger than MNPs.

3. FTIR analysis of FA-MNPs indicated the presence of FA conjugated to PEG-

MNPs.

4. 505 μM doxorubicin loaded to FA-MNPs was determined after 24 h mixing of

FA-MNPs and doxorubicin. 82.27. 75.66 and 114.20 μM doxorubicin released

from Dox-FA-MNPs was detected by measurement of absorbance of

supernatant at 480 nm after 96 h. Acidity of environment increased the drug

release rate when compared the drug releases in two different pH values of

acetate buffer. Components of buffer could affects the release of drug compared

the drug releases in acetate buffer and dH2O.

5. Doxorubicin resistant HeLa (HeLa/Dox) cells were developed from parental

HeLa cells. HeLa/Dox was 37 times resistant to doxorubicin when compared to

HeLa cells according to their IC50 values.

58

6. FA-MNPs and Dox-FA-MNPs were internalized by HeLa cells and

Internalization of them was detected by purssian blue staining and by

fluorescence of doxorubicin.

7. FA-MNPs were not cytotoxic to HeLa and HeLa/Dox cells. Dox-FA-MNPs

effectively showed antiproliferative activity on HeLa cells while they did not

show same effect on HeLa/Dox cells.

Doxorubicin loaded folic acid conjugated PEG coated magnetic nanoparticles is

effective to kill sensitive cancer cells and may result in decrease in the side effects

of doxorubicin.

59

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APPENDIX A

BUFFERS AND SOLUTIONS

A.1. Freezing medium

9ml FBS (Biochrome, Germany)

1ml DMSO (Applichem, Germany)

Mixed and stored at +4C

A.2. Phosphate buffered saline (pH 7.2)

1 PBS tablet (Sigma. Germany) in 200ml distilled water.

After the tablet dissolved PBS is autoclaved at 121C for 20 minutes.

A.3. 4X Tris buffer (pH 7)

30.35 g Tris base (Bioshop)

pH is adjusted to 7. Then, volume is completed to 500 mL with distilled water.

A.4. Acetate buffer (pH 4.13. 100 mL)

0.4843 g acetic acid (Sigma)

0.2633 g sodium acetate trihydrate (Sigma)

100 ml water.



0.9669 g sodium acetate (Sigma)

100 ml water.



1.3578 g sodium acetate (Sigma)

100 ml water.

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APPENDIX B

ATR AND FTIR SPECTRA

B.1. Attenuated Total Reflection (ATR) Spectrum of PEGmonooleate

Figure B. 1: ATR Spectrum of PEGmonooleate

The FTIR analyses of PEGmonooleate showed 3 characteristic peaks. The peaks at

946 cm-1

and 1109 cm-1

indicated the stretching vibration of functional CH2 group and

the stretching vibration of functional group of C–O of PEG respectively while the

absorption band observed at 1735 cm-1

was caused by C=O carbonyl group of PEG.

0

50

100

150

200

250

0 500 1000 1500 2000 2500 3000 3500 4000

Tran

smit

tan

ce (

%)

Waveleght (cm-1)

945

1109

1735

68

B.2. Fourier Transform Infrared Spectroscopy (FTIR) Spectrum of Folic Acid

Figure B. 2: FTIR Spectrum of Folic Acid.

The FTIR result showed the characteristic absorption peaks 1433, 1606 and 1695 cm-1

of FA. The characteristic band of 1484 and 1411 cm-1

were belonging to the phenyl ring

of folic acid. 1606 cm-1 band indicated –NH2 bending vibration of FA (Keskin, 2012).

0

20

40

60

80

100

120

300800130018002300280033003800

Tran

smit

tan

ce (

%)

Waveleght (cm-1)

1695 1606 1484

69

APPENDIX C

CYTOTOXITY OF FA-MNPs ON HeLa AND HeLa/Dox CELL LINES

C.1. HeLa Cell Line

0 38 50 67 89 119

158

211

281

375

500

0

50

100

150

ns ns * * * ***

***

***

***

***


% C

ell P

rolife

rati

on

Figure C. 1: Antiproliferative effects of FA-MNPs on HeLa cell line. Results were

obtained from three independent experiments. represented as mean SEM.

70

C.2. HeLa/Dox Cell line

0 38 50 67 89 119

158

211

281

375

500

0

50

100

150

ns ns* ** **

*

***

***

***

***


% C

ell P

rolife

rati

on

Figure C. 2: Antiproliferative effects of FA-MNPs on HeLa/Dox cell line. Results

were obtained from three independent experiments. represented as mean SEM.

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APPENDIX D

STANDARD CURVE OF DOXORUBICIN

Figure D. 1: Representative standard curve of Doxorubicin.

y = 0,0126x R² = 0,999

0

5

10

15

20

25

30

35

40

45

50

0 1000 2000 3000 4000

Ab

sorb

ance

at

48

0 n

m

Doxorubicin Concentration (μM)

Standard Curve of Doxorubicin

73

APPENDIX E

CONCENTRATION OF DOXORUBICIN LOADED TO FA-MNPs

Figure E. 1: Concentrations of Doxorubicin that was loaded to FA-MNPs in

different concentrations of Doxorubicin.

0

100

200

300

400

500

600

0 500 1000 1500 2000

Load

ed

Dru

g C

on

c (μ

M)

Total Drug Conc (μM)

SYNTHESIS AND CHARACTERIZATION OF ...Co-supervisor: Assist. Prof. Dr. Serap Yalçın Azarkan August...

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